Functional endothelin/sarafotoxin receptors in the rat uterus

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) families were detected in the rat uterus. These receptors specifically bind 125I-SRTX-b (Bmax = 220 fmol/mg protein), as well as ET-1, ET-3 and SRTX-c (IC50's 10, 5, 300 and 780 nM, respectively). Activation of the uterine ET/SRTX receptors induced dose-dependent phosphoinositide (PI) hydrolysis and three typical contractile responses: 1) increase in the muscle tonic tension; 2) increase in frequency of the spontaneous rhythmic contractions; 3) decrease of relaxation in each spontaneous rhythmic cycle. All three effects appeared at doses as low as 0.5-1 nM. Dose responses yield ED50 values of 5.5, 30 and 680 nM for ET-1, SRTX-b and ET-3, respectively. SRTX-c was the least effective peptide in achieving decrease in relaxation. In view of these results, and since the uterine responses to the peptides were almost immediate and reversible, we suggest that the functional ET/SRTX receptor of the rat uterus that is coupled to PI hydrolysis may be of physiological significance.     

Functional endothelin/sarafotoxin receptors in rat heart myocytes: structure-activity relationships and receptor subtypes

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) family were characterized in newborn rat heart myocytes using human and rat endothelins (ET-1 and ET-3, respectively), SRTX-b and SRTX-c. Binding studies in intact cells and homogenates revealed significantly higher affinities of ET-1 and SRTX-b than of ET-3 and SRTX-c towards these receptors. This binding profile of ET/SRTX peptides points to their interaction with the receptor subtype designated E-S alpha. All four peptides induced time- and dose-dependent phosphoinositide hydrolysis with the following rank order of potency: ET-1 greater than SRTX-b greater than SRTX-c greater than ET-3. Thus, ET-3 which possesses an intermediate affinity toward the receptor was the least effective with regard to this response. These results confirm and extend our earlier report that the ET/SRTX peptides interact with a newly characterized receptor(s) associated with phosphoinositide metabolism and Ca2+ mobilization. The initiation of inositol phosphate formation is largely independent of extracellular Ca2+, verapamil and nifedipine, indicating that the ET/SRTX peptides are not agonists for the voltage-dependent Ca2+-channels.     

Endothelin and sarafotoxin: influence on steroid-regulated motility of rat uterus.

The sarafotoxins (SRTX) and endothelins (ET) were shown to influence the motility of the isolated rat uterus by inducing an increase in the rate and in the maximum tension of the spontaneous rhythmic contractions and a suppression of the relaxation phase of these contractions. Ovariectomized rats, 24 weeks post-operation, show no spontaneous motility of their uteri and the SRTX/ET peptides induce only a slight tonic increase in the uterine tension. Treatment with 17 beta estradiol restores spontaneous motility and sensitivity to the SRTX/ET peptides in all three contraction modes. It is concluded that the influence of the SRTXs and ETs on uterine motility depends on the hormonal status of the animal.     

Structure-Function Relationships of Endothelins, Sarafotoxins, and Their Receptor Subtypes

The endothelins (ETs) and sarafotoxins (SRTXs) are two structurally related families of potent vasoactive peptides. Although their physiological functions have yet to be precisely elucidated, it seems likely that the ETs are involved in pathophysiological conditions such as hypertension and heart failure. In this minireview, recent advances in the biochemical characterization of the ET/SRTX system, with special reference to structure-function relationships and ET/SRTX receptor subtypes, are described, as well as the recent cloning and expression of ET receptors.     

The endothelin/sarafotoxin-induced increase of the proliferation of undifferentiated and DMSO-differentiated GEM-81 goldfish erythrophoroma cells is mediated by ETB receptors

Endothelins (ETs) and sarafotoxins (SRTXs) have been reported to exert ET(B)-mediated effects on vertebrate pigment cells. GEM-81 cell line, a red pigment cell-derived cutaneous tumor of the teleost Carassius auratus, expresses ET(B) receptors and can be differentiated with 1.5% DMSO treatment, thus constituting an useful model to investigate ET and SRTX effects on cultured fish pigment cells. Our aim was to characterize the pharmacology and biological effects mediated by ET receptors in DMSO-differentiated and undifferentiated cells. ET subtype receptors and their respective Ki values in both cell types were determined by competitive binding assays using (125)I ET-1 and BQ-485 (an ET(A) antagonist) or BQ-788 (an ET(B) antagonist). BQ-788, but not BQ-485, significantly reduced (125)I-ET-1 binding in both cell types, with similar low (Ki > nM) affinities. To determine the proliferation effects of ETs/SRTXs, cells were treated for 72 h with the hormones, and counted in a hemocytometer. The proliferation assays were repeated for SRTX S6c in the presence or absence of BQ-788. The results demonstrated that, with the exception of ET-1 (biphasic effect) and ET-3 (no significant effect) in undifferentiated GEM-81 cells, all the tested hormones induced increases in the proliferation of both types of cells. The hormones were equipotent in DMSO-differentiated cells, which exhibited increased sensitivity to ETs, but not to SRTXs, as compared with undifferentiated cells. The BQ-788 antagonistic effect was also exerted on the proliferation responses to SRTX S6c. These results corroborate the long and important evolutionary history of the ET/SRTX receptor system in vertebrate pigment cells.     

The Endothelin/Sarafotoxin‐Induced Increase of the Proliferation of Undifferentiated and DMSO‐Differentiated GEM‐81 Goldfish Erythrophoroma Cells is Mediated by ETB Receptors

Endothelins (ETs) and sarafotoxins (SRTXs) have been reported to exert ET_B‐mediated effects on vertebrate pigment cells. GEM‐81 cell line, a red pigment cell‐derived cutaneous tumor of the teleost Carassius auratus, expresses ET_B receptors and can be differentiated with 1.5% DMSO treatment, thus constituting an useful model to investigate ET and SRTX effects on cultured fish pigment cells. Our aim was to characterize the pharmacology and biological effects mediated by ET receptors in DMSO‐differentiated and undifferentiated cells. ET subtype receptors and their respective Ki values in both cell types were determined by competitive binding assays using ¹²⁵I ET‐1 and BQ‐485 (an ET_A antagonist) or BQ‐788 (an ET_B antagonist). BQ‐788, but not BQ‐485, significantly reduced ¹²⁵I‐ET‐1 binding in both cell types, with similar low (Ki > nM) affinities. To determine the proliferation effects of ETs/SRTXs, cells were treated for 72 h with the hormones, and counted in a hemocytometer. The proliferation assays were repeated for SRTX S6c in the presence or absence of BQ‐788. The results demonstrated that, with the exception of ET‐1 (biphasic effect) and ET‐3 (no significant effect) in undifferentiated GEM‐81 cells, all the tested hormones induced increases in the proliferation of both types of cells. The hormones were equipotent in DMSO‐differentiated cells, which exhibited increased sensitivity to ETs, but not to SRTXs, as compared with undifferentiated cells. The BQ‐788 antagonistic effect was also exerted on the proliferation responses to SRTX S6c. These results corroborate the long and important evolutionary history of the ET/SRTX receptor system in vertebrate pigment cells.     

Endothelin/sarafotoxin receptor heterogeneity: evidence for different glycosylation in receptors from different tissues.

Neuraminidase was used in an attempt to determine whether the endothelin (ET)/sarafotoxin (SRTX) receptor subtypes are glycoproteins and, if so, to determine the role of the carbohydrate moiety in the binding of ligands to the receptor. Incubation of rat cerebellar membranes with neuraminidase was accompanied by a decrease in the capacity of the receptors to bind ET-1 and SRTX-b. In contrast, treatment of the rat caudate putamen and strium or of guinea pig ileum with the enzyme did not affect the binding properties of these receptors. Following exposure of [125I]-ET-1 affinity-labeled receptor to neuraminidase, gel electrophoresis and autoradiography revealed a decrease in molecular mass in the cerebellar and atrial preparations of about 2.5-2.8 kDa. These data indicate that some of the ET/SRTX receptors are glycoproteins and that the sugar moiety is important for ligand binding. Thus, glycosylation might be responsible for the observed heterogeneity among ET/SRTX receptors.     

Sarafotoxin expression in the bronchopulmonary tract: immunohistochemical occurrence and colocalization with endothelins

The immunohistochemical occurrence of sarafotoxin (SRTX), a snake venom peptide under strong evolutionary control, was investigated in the pulmonary diffuse neuroendocrine system (PDNES) of newborn cats and rats. By applying the avidin-biotin-peroxidase complex method on serial lung sections, we have demonstrated its distribution and colocalization with different endothelin (ET) isoforms. A light microscopic study revealed apparent immunostaining for SRTX in neuronal components and smooth muscle tissue and in neuroepithelial bodies (NEB), while isolated neuroendocrine cells (NEC) remain unlabelled. Comparison of the SRTX reactivity pattern with that of different ET peptides on adjacent lung sections showed colocalization of SRTX-b with ET-3 in NEB, intrapulmonary ganglion cells and nerve fibres, on the one hand, and with ET-1 in airway and vascular smooth muscle cells, on the other. These findings, in addition to the remarkable functional and structural similarities between SRTX and ET peptides, suggest a common evolutionary origin and biological significance of sarafotoxin and endothelins. Moreover, this is the first time that a toxic peptide has been demonstrated in the PDNES.     

Transforming growth factor beta-1 attenuates endothelin-1-induced functions in neonatal cardiac myocytes

In the present study we characterized a "crosstalk" mechanism between transforming growth factor beta-1 (TGF beta-1) and endothelin-1 (ET1) signaling pathways in neonatal cardiac myocytes. A 5 minute pretreatment with 1 ng/ml concentrations of TGF beta-1 attenuated ET1-induced negative chronotropic effects and translocation of the alpha, delta and varepsilonPKC isozymes to the particulate cell fraction. We found no effect of TGF beta-1 on responses induced by the P(2) purinergic agonist ATP or phorbol ester. Treatment of cardiac myocytes with acidic fibroblast growth factor (aFGF) did not alter ET1- or ATP-mediated effects on contraction rate or translocation of PKC isozymes to the particulate fraction. Our studies suggest that TGF beta-1 may act as a negative modulator of ET1- but not ATP- or phorbol ester-induced PKC isozyme signaling events in neonatal cardiac myocytes. A better understanding of the complex ET1 and TGF beta-1 signaling mechanisms in neonatal heart cells should enhance our knowledge regarding the interplay between these pathways.     

Chromatic effects of endothelin family peptides in non-innervated fish, Synbranchus marmoratus, melanophores

The biological activity of endothelins (ETs) in non-innervated Synbranchus marmoratus melanophores was demonstrated. These peptides induced a dose-dependent pigment aggregation (lightening skin) in these cells. However, they presented EC50's (effective concentration required to produce 50% of response) 26, 106 and 35 times higher than, respectively, the melanin concentrating hormone (MCH) EC50, and exhibited a characteristic temporal and dose-dependent autodessensibilization of the aggregative effect on the melanophores of this fish.The receptor characterization suggested the presence of the ET(B) subtype, since BQ-788 (selective antagonist of ET(B)) but not BQ-485 (selective antagonist of ET(A)) blocked the aggregative effect of the hormones. Confirming these data, sarafotoxin (SRTX) S6c, a toxin selective for ET(B), induced maximal aggregation of pigment granules. S6c presented an EC50 6.8 times higher than the MCH EC50, and 3.9, 15.6 and 5.1 times lower than the EC50's ETs, respectively.The melanotropic effect of SRTX S6b and vasoactive intestinal contractor (VIC) were demonstrated for the first time in this work. SRTX S6b induced a dose-dependent pigment aggregation and presented an EC50 2.54 and 17.2 times higher than the S6c and MCH EC50's, respectively. Compared to the ETs it was 1.53, 6.19 and 2.03 times lower, respectively.     

Cardiac α1-Adrenoceptors that regulate contractile function: Subtypes and subcellular signal transduction mechanismsthat regulate contractile function: Subtypes and subcellular signal transduction mechanisms

Activation of α₁-adrenoceptors as well as endothelin (ET) and angiotensin II (Ang II) receptors in cardiac muscle is coupled to acceleration of the hydrolysis of phosphoinositide (PI), with resultant production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol. There is an excellent correlation between the extent of acceleration of the PI hydrolysis and the positive inotropic effect (PIE) under most experimental conditions after the administration of α-adrenoceptor agonists, ET and Ang II in the rabbit ventricular muscle. The PIE of the α-adrenoceptor agonists, ET and Ang II is associated with a negative lusitropic effect and an increase in the sensitivity of myofilaments to Ca²⁺ ions. The PIE can be selectively inhibited by inhibitors of protein kinase C (PKC) such as staurosporine, NA 0345 and H-7, with little effect on the PI hydrolysis and the PIE of isoproterenol and Bay k 8644. Surprisingly, an activator of PKC, phorbol 12,13-dibutyrate (PDBu), selectively and more completely inhibited the PIE and acceleration of PI hydrolysis induced by the α-adrenoceptor agonists as well as by ET and Ang II in the rabbit. These receptor agonists consistently cause intracellular alkalinization by activation of Na⁺−H⁺ exchange, while the effects on membrane ion channel activities are divergent. For example, α-adrenoceptor agonists cause monophasic prolongation of the action potential, the time course of which coincides well with that of the PIE, while ET and Ang II produce a biphasic change in action potential duration, i.e., the long-lasting prolongation preceded by a transient abbreviation. α-Adrenoceptor agonists scarcely affect I_Ca, whereas ET elicits a biphasic alteration of the current. In addition, the potassium current, I_Kl, is markedly suppressed by α-adrenoceptor agonists, but this effect is not revealed with Ang II under the same experimental condition. These results indicate that the effects of α₁-adrenoceptors stimulation are partially shared by those of ET and Ang II receptor activation in the heart. Approximately 60% of the total population of α₁-adrenoceptors in the rabbit ventricle are composed of α_1B subtype, which is susceptible to chlorethylclonidine (CEC) and is predominantly responsible for the α₁-mediated PIE and PI hydrolysis. The remaining fraction that belongs to α_1A-adrenoceptors subtype is further subclassified into the WB 4101-sensitive (partly coupled to PI hydrolysis) and the niguldipinesensitive (PI hydrolysis-unrelated) subtypes. Special issue dedicated to Dr. Kinya Kuriyama.     

Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart

Impulse-conducting Purkinje fibers differentiate from myocytes during embryogenesis. The conversion of contractile myocytes into conduction cells is induced by the stretch/pressure-induced factor, endothelin (ET). Active ET is produced via proteolytic processing from its precursor by ET-converting enzyme 1 (ECE1) and triggers signaling by binding to its receptors. In the embryonic chick heart, ET receptors are expressed by all myocytes, but ECE1 is predominantly expressed in endothelial cells of coronary arteries and endocardium along which Purkinje fiber recruitment from myocytes takes place. Furthermore, co-expression of exogenous ECE1 and ET-precursor in the embryonic heart is sufficient to ectopically convert cardiomyocytes into Purkinje fibers. Thus, localized expression of ECE1 defines the site of Purkinje fiber recruitment in embryonic myocardium. However, it is not known how ECE1 expression is regulated in the embryonic heart. The unique expression pattern of ECE1 in the embryonic heart suggests that blood flow-induced stress/stretch may play a role in patterning ECE1 expression and subsequent induction of Purkinje fiber differentiation. We show that gadolinium, an antagonist for stretch-activated cation channels, downregulates the expression of ECE1 and a conduction cell marker, Cx40, in ventricular chambers, concurrently with delayed maturation of a ventricular conduction pathway. Conversely, pressure-overload in the ventricle by conotruncal banding results in a significant expansion of endocardial ECE1 expression and Cx40-positive putative Purkinje fibers. Coincident with this, an excitation pattern typical of the mature heart is precociously established. These in vivo data suggest that biomechanical forces acting on, and created by, the cardiovascular system during embryogenesis play a crucial role in Purkinje fiber induction and patterning.     

内皮素对离体大鼠心肌细胞的影响

本文应用离体成年大鼠心肌细胞,研究内皮素作用的细胞机制,发现10~(-9)—10~(-7)mol/L内皮素可引起心肌细胞挛缩、胞浆乳酸脱氢酶漏出和细胞总钙量增加,且具有剂量-效应关系。内皮素可促进~(45)Ca内流,其作用为钙通道阻断剂维拉帕米所拮抗。预先用负载荧光染料Fura-2的心肌细胞与内皮素孵育,可见胞浆游离钙显著增加,其作用亦可为维拉帕米抑制。结果提示:内皮素主要通过电位依赖的钙通道促进心肌细胞Ca~(2+)内流,产生其生物学效应。     

The endothelin ETB receptor mediates both vasodilation and vasoconstriction in vivo.

It has been suggested that the endothelin (ET) ETB receptor could mediate endothelium-dependent vasodilation to ET-1 or ET-3, but its in vivo role is still largely unknown. We used sarafotoxin S6C, a selective agonist of the ETB receptor, to study the in vivo effects of ETB stimulation. SRTX S6C induced a transient decrease in blood pressure, followed by a long-lasting pressor response accompanied by a marked renal and mesenteric vasoconstriction. No constriction was observed in isolated mesenteric arteries in vitro, indicating that the in vivo vasoconstrictor effect is most likely indirect. The pressor effect of SRTX S6C was not dependent on central stimulation of ETB receptors and was not mediated by catecholamines from the adrenal medulla, prostanoids or ET-1.     

Pharmacological and structural characterization of long-sarafotoxins, a new family of endothelin-like peptides: Role of the C-terminus extension

Long-sarafotoxins (l-SRTXs) have recently been identified in both the venom of Atractaspis microlepidota and that of Atractaspis irregularis. They are characterized by different C-terminus extensions that follow the invariant Trp21, which plays a crucial role in endothelin-receptor binding. We initially determined the toxicity and three-dimensional structures of two chemically synthesized l-SRTXs that have different C-terminus extensions, namely SRTX-m (24 aa, including extension "D-E-P") and SRTX-i3 (25 aa, including extension "V-N-R-N"). Both peptides were shown to be highly toxic in mice and displayed the cysteine-stabilized α-helical motif that characterizes endothelins and short-SRTXs, to which a longer C-terminus with variable flexibility is added. To discern the functional and pharmacological consequences of the supplementary amino acids, different chimerical as well as truncated forms of SRTX were designed and synthesized. Thus, we either removed the extra-C-terminal residues of SRTX-m or i3, or grafted the latter onto the C-terminal extremity of a short-SRTX (s-SRTX) (ie. SRTX-b). Our competitive binding assays where SRTXs competed for iodinated endothelin-1 binding to cloned ET(A) and ET(B) receptor subtypes over-expressed in CHO cells, revealed the essential role of the C-terminus extensions for ET-receptor recognition. Indeed, l-SRTXs displayed an affinity three to four orders of magnitude lower as compared to SRTX-b for the two receptor subtypes. Moreover, grafting the C-terminus extension to SRTX-b induced a drastic decrease in affinity, while its removal (truncated l-SRTXs) yielded an affinity for ET-receptors similar to that of s-SRTXs. Furthermore, we established by intracellular Ca(2+) measurements that l-SRTXs, as well as s-SRTXs, display agonistic activities. We thus confirmed in these functional assays the major difference in potency for these two SRTX families as well as the crucial role of the C-terminus extension in their various pharmacological profiles. Finally, one of the chimeric toxin synthesized in this study appears to be one of the most potent and selective ligand of the ET(B) receptor known to date.     

Potentiation by Ca2+ ionophores and inhibition by extracellular KCl of endothelin-induced phosphoinositide turnover in C6 glioma cells.

Interactions between endothelin-1 (ET)-induced phosphoinositide (PI) hydrolysis and agents that increase Ca2+ influx (i.e. A23187 and ionomycin) or induce depolarization (i.e. KCl) were investigated using C6 glioma. A23187 dose-dependently potentiated ET (30 nM)- and ATP (100 microM)-induced [3H]inositol phosphate (IP) accumulation. This potentiation was associated with an increase in the maximal stimulation elicited by both ET and ATP but their EC50 values were unchanged. This effect of A23187 occurred at concentrations that did not affect basal PI turnover; i.e. 10 nM-3 microM. Ionomycin within the range of 1 nM-1 microM also significantly enhanced ET-induced PI breakdown and this effect was associated with an increase of [Ca2+]i. KCl in a concentration-dependent manner (14.7-54.7 mM) markedly inhibited PI breakdown elicited by ET and ATP, but had much less inhibition on basal activity and no effect on A23187- and ionomycin-induced responses. In parallel, KCl added before or after ET, sharply attenuated the increase of ET-induced [Ca2+]i but did not affect basal level or ionomycin-induced [Ca2+]i response. Neither the potentiation by A23187 nor the inhibition by KCl of ET-induced PI turnover was observed in cultured cerebellar astrocytes. Our results suggest that the cell type-specific regulation by Ca2+ ionophores and KCl on ET-induced PI metabolism is closely related to perturbation of [Ca2+]i.     

Characterization and localization of a novel neuroreceptor for the peptide sarafotoxin

We have recently shown that the rat atrium and brain contain specific high affinity receptors for the novel snake vasoconstrictor peptide sarafotoxin-b (SRTXb), and demonstrated toxin-induced phosphoinositide hydrolysis. Here we report on the characteristics of 125I-SRTXb receptors and their regional distribution in rat brain. 125I-SRTX receptors in the rat brain bind the toxin rapidly and with high affinity. The binding was not inhibited by ligands of known neurotransmitter receptor and ion channels. 125I-SRTX receptors have a distinctive regional distribution. The highest densities were observed in the cerebellum, thalamus and hypothalamus (850, 550 and 450 fmol/mg protein, respectively) and the lowest densities in the caudate and cerebral cortex (82 and 62 fmol/mg protein, respectively). Taken together our results suggest that mammalian brains contain a hitherto undetected neuroreceptor that may operate in neurotransmission with a "SRTX-like" brain peptide, similar to the SRTX homologous vasoconstrictor peptide of the mammalian endothelium endothelin.     

Comparative studies of phosphoinositide hydrolysis induced by endothelin-related peptides in cultured cerebellar astrocytes, C6-glioma and cerebellar granule cells

Effects of endothelin (ET) homologues (ET-1, 2, 3 and sarafotoxin S6b) and its precursor (big ET-1) on phosphoinositide (PI) turnover were compared in neurally-related cell cultures. All ET-related peptides induced a robust increase of PI turnover in cerebellar astrocytes, C6-glioma and cerebellar granule cells. The rank order of potency in stimulating PI turnover was ET-1 = ET-2 greater than or equal to S6b greater than ET-3 greater than big ET-1 for granule cell neurons, while it was ET-1 greater than or equal to ET-2 greater than or equal to S6b greater than big ET-1 greater than ET-3 for astrocytes and C6-glioma cells. Short-term pretreatment with phorbol dibutyrate (PDBu) attenuated the ET-1-induced PI response in all three types of cultures. However, long-term pretreatment with PDBu attenuated the response in granule cells and C6-gliomas, but enhanced responses to ET and ATP in astrocytes. Long-term exposure of cells to pertussis toxin (PTX) attenuated the PI response to ET in astrocytes and C6-gliomas, but not in granule cells. Thus, phospholipase C-coupled ET receptors are expressed in both neurons and glial cells, but they differ considerably in their pharmacological selectivity and signal transduction mechanisms in stimulating PI hydrolysis.     

Insulin provokes co-ordinated increases in the synthesis of phosphatidylinositol, phosphatidylinositol phosphates and the phosphatidylinositol-glycan in BC3H-1 myocytes.

BC3H-1 myocytes were cultured in the presence of [3H]inositol or [3H]glucosamine during their entire growth cycle to ensure that all lipids containing inositol and glucosamine were labelled to isotopic equilibrium or maximal specific radioactivity. After such labelling, a lipid (or group of lipids), which was labelled with both inositol and glucosamine, was observed to migrate between phosphatidylinositol 4-phosphate and phosphatidylinositol (PI) in two different t.l.c. systems. Insulin provoked rapid, sizeable, increases in the inositol-labelling of this lipid (presumably a PI-glycan), and these increases were similar to those observed in PI and PI phosphates. Our results indicate that insulin provokes co-ordinated increases in the net synthesis de novo of PI and its derivatives, PI phosphates and the PI-glycan, in BC3H-1 myocytes. This increase in synthesis of PI may serve as the mechanism for replenishing the PI-glycan during stimulation of its hydrolysis by insulin. Moreover, increases in the content of the PI-glycan may contribute to increases in the generation of head-group 'mediators' during insulin action.     

Metabolic regulation of coronary vascular tone: role of endothelin-1

Coronary tone is determined by a balance between endogenously produced endothelin and metabolic dilators. We hypothesized that coronary vasodilation during augmented metabolism is the net result of decreased endothelin production and increased production of vasodilators. Isolated rat myocytes were stimulated at 0, 200, and 400 beats/min to modify metabolism. Supernatant from these preparations was added to isolated coronary arterioles with and without blocking vasoactive pathways (adenosine, bradykinin, and endothelin). Chronically instrumented swine were studied while resting and running on a treadmill before and after endothelin type A (ET(A)) receptor blockade. The vasodilatory properties of the supernatant increased with increased stimulation frequencies. Combined blockade of adenosine and bradykinin receptors abolished vasodilation in response to supernatant of stimulated myocytes. ET(A) blockade increased vasodilation to supernatant of unstimulated myocytes but did not affect dilation to supernatant of myocytes stimulated at 400 beats/min. In vivo, ET(A) blockade resulted in coronary vasodilation at rest, which waned during exercise. Thus endothelin has a tonic constrictor influence through the ET(A) receptor at low myocardial metabolic demand but its influence decreased during increased metabolism.